Caky chromatographic column and the method for producing it and its applications

ABSTRACT

The present invention discloses a new and improved kind of chromatographic cake and its manufacturing methods and applications. The objective of this invention is to provide an improved chromatographic apparatus and methods for manufacturing such an apparatus, whereby the separation and simultaneous renaturation and purification of biopolymers can be efficiently performed with excellent results. The chromatographic cakes of this invention generally comprise a chromatographic packing cake with a mobile phase inlet and a mobile phase outlet, together with a chromatographic packing packed into an inner cavity region of the chromatographic packing cake. The ratio of the thickness to the diameter of the inner cavity region of the chromatographic packing cake is less than or equal to 1. A method of manufacturing such chromatographic cakes generally comprises the steps of: 1) Manufacturing the chromatographic packing cake, wherein the chromatographic packing cake includes combined upper and lower clamp plates having, respectively, a mobile phase inlet or outlet, and a cake body with at least one lateral hole or aperture; wherein the ratio of the thickness to the diametral dimension of the inner cavity of the chromatographic packing cake is smaller than or equal to 1; and, 2) adding chromatographic packing in whole or at least in part using the lateral hole(s) of the chromatographic packing cake to fill the inner cavity of the chromatographic-packing cake with a suitable packing material.

REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/CN20/00333 filed May 16, 2002.

TECHNICAL FIELD

This invention relates to a kind of chromatographic cake and itsmanufacturing method and application, especially to a kind ofchromatographic cake and its manufacturing method and application whichis applicable to biopolymer separation or renaturation with simultaneouspurification.

BACKGROUND OF THE INVENTION

Chromatography and capillary electrophoretic methods are two means usedfrequently for the separation of biopolymers. Because of the limitationof the quantity of the employed mobile phase in capillaryelectrophoretic method, however, such methods can generally only be usedon an analytical scale. Chromatography is now the most important and themost effective separation method for the separation, renaturation andpurification of biopolymers. It would be desirable to be able to usesuch technique not only for analytical scale, but also for productionscale.

When chromatography is used for separation, it is commonly accepted thatthe separating effect is proportional to the number of column plates,e.g., column length. If the column is too long, however, it is tooexpensive and it leads to a higher column pressure, which must becontrolled with a high performance liquid chromatograph. It is reportedin the literature that evidence can be obtained showing effectivebiopolymer separation with a short column using the stoichiometricdisplacement model for solute-in-liquid chromatography; and, a packedcolumn of 2 mm in length using a slice of membrane having a thickness ofabout 2 mm that is cut off from a continuous rod is used to makebiopolymer separations with good results when used on an analyticalscale. It is still unknown, however, whether such a short column withstable effects can be utilized for industrialized production, as well asto what degree such a column can be shortened and still be effective.

At present, a column for high performance liquid chromatography employedat a production scale level is generally packed with particles having adiameter of about 20–30 μm. It is ideal that the ratio of column lengthto diameter be about 10 or less to avoid a too-high column pressureunder conditions of high flow rate, thereby causing decreasingseparation effects. In order to ameliorate the fact that the volume ofthe column bed always becomes effectively smaller under the pressureaccumulated by the soft matrix over an increased column length,Pharmacia Co., which is famous around the world for its production ofchronographic media adapted for use at low and middle pressureconditions, has offered a cake-shaped chromatographic column withshorter thickness (but having a length of at least 10 cm) and of greaterdiameter. When in use, several cake-shaped chromatographic columns ofthis type would typically be connected together in series. Therefore,the sum of the lengths of these serially connected columns is still manytimes greater than the diameter of an individual cake-shaped column, sothe flow rate through this series of chromatographic cakes still cannotbe too high when it is applied.

Many proteins expressed with E. coli in biotechnology exist in the formof inclusion body in E. coli because of its high hydrophobicity.Although the primary structures of such proteins may be correct, theirthird or fourth structures are basically wrong. As the inclusion bodygenerally has the property of high hydrophobicity, it should bedissolved with a denaturant at high concentration, such as 7.0 mol/Lguanidine hydrochloride (GuHCl) or 8.0 mol/L urea. Then the renaturationof the proteins can be processed. In the current technology, thedialysis method and the dilution method are commonly used for proteinrenaturation. Nevertheless, these two methods not only have a lowrenaturation effect (on the order of about 5%–20% generally), but theyalso require too much time resulting in failure to realize the objectiveof separating impure proteins. One alternative technology has beendeveloped in which a denatured protein is renatured and simultaneouslypurified by high performance hydrophobic interaction chromatography(HPHIC). However, if some precipitates of proteins occur by sampleinjection, the chromatographic column in this technique will be blockedor damaged. Thus, the denatured facilities currently used for proteinrenaturation and purification and the multifunctional protein-purifyingunit as described above have major limitations which reduce theirutility and effectiveness.

It can be concluded that the following major problems currently exist inthe separation and purification of proteins in various biotechnologyprocesses:

1. Separation and purification of biopolymers made in a glass column, aplastic column or a stainless steel column, packed by soft based media,and having large diameters. The shortcomings of these processes includelow column efficiency, need for large volumes of media, high consumptionof mobile phase, and long production periods.

2. When small solutes are separated with a chromatographic column, theresolution should be proportional to the column length. The ratio ofcolumn diameter to column length is normally about 1:10. The columnlength has a small effect on the resolution of biopolymers. In general,a column with a length of about 5 cm is selected. When such a column ispacked with small particles, the chromatographic system through pressureis obviously increased. Such a column should therefore be used with ahigh-pressure liquid chromatograph, but this results in increasedproduction costs. Such increased costs counterbalance some of theadvantages of using a column packed by small particles, namely highefficiency, large volume and good reproducibility.

3. For the usual chromatographic columns, it is preferred that sampleshaving a high viscosity not enter the columns, and that no more than asmall quantity of precipitates from samples are allowed to settle in thecolumn head. In practice, however, the inlet and outlet conditions ofthe mobile phase cannot readily be changed, so such control is notconvenient for the operation.

4. In a usual separation and purification process, relatively pureproducts can be obtained only through the sequential steps ofrenaturation, removal of denaturants, coarse purification, and multistepfine purification. These steps represent a long and cumbersomeprocessing technology usually involving great loss of mass andbioactivity and, thus, with a relatively low recovery (generally nohigher than about 5–20%).

5. Common renaturation methods include a dilution method and a dialysismethod. With the dilution method, many dilution steps should be taken togradually decrease the concentration of the denaturing agent employed.Thus, such a process brings many handling difficulties for the laterseparation and purification steps if, at each stage, samples are dilutedtens, or even hundreds, of times. The dialysis method, on the otherhand, takes too much time (e.g., 24 hours for only one dialysis stepgenerally), and, furthermore, the dialysis agent should be changed manytimes. In addition, with the above two renaturation methods, the subjectproteins are easily aggregated and precipitated resulting in a longrenaturation time during the renaturation process.

6. There are many steps involved in the current separation andpurification methods. In the course of such separation and processingsteps, the volume of solution containing the subject proteins is alwaysincreasing. Also, each step of these methods requires substantialassociated processing equipment. Therefore, current separation andpurification techniques require a relatively large investment inequipment with correspondingly high production costs.

At present, for packing and manufacturing a chromatographic column,satisfactory column efficiency can be obtained when chromatographicpacking material is packed using familiar axial and radialpressurization techniques. But these two methods are only applicable tothe packing process for a relatively long chromatographic column whereinthe ratio R of column length to diameter is greater than unity (i.e.,R>1). There are no ideal packing and manufacturing methods currentlyavailable, however, for the process of packing a chromatographic “cake”wherein the ratio R of cake thickness to cake diameter is smaller thanor equal to unity (i.e., R≦1). If the traditional technology were usedto pack a chromatographic cake wherein R≦1, the following problems wouldlikely occur:

1. In the traditional process to pack a chromatographic column, liquidgoes through the column inlet and column outlet in the axial direction(i.e., along the axis of the column). In general, the traditionalpacking processes are only applicable to a chromatographic columnwherein the ratio R of column length to column diameter is greater thanunity.

2. Because the technique of packing columns properly requires a highlevel of skill, if an operator is lacking the necessary skills,circumstances may occur in which a column is not packed properly, forexample having a tight outlet and/or a loose inlet. An improperly packedcolumn has low reproducibility of results because of the defect in thecolumn packing.

3. In a conventional chromatographic column packing process, the inletend of the column can be made even and smooth with a blade after thecolumn has been packed. For a chromatographic cake, however, there areno easy and effective methods to make such a large surface area of thepacking, such as the inlet end of the column, even and smooth afterpacking the cake.

4. In general, a chromatographic column needs to be repacked after longuse at high pressure. Before a column is repacked, the sunken inlet endof a chromatographic column is typically repaired to prolong columnlife. In order for the sunken inlet end of a chromatographic column tobe repaired, however, the column head must be dismantled to remove thefrit. For a column with a relatively small diameter, it is generallyeasy to remove the frit because of the small surface area of the frit.But, the surface area of the inlet end of a “chromatographic cake” isgenerally more than one up to 100 times or more greater than that of acomparable conventional chromatographic column. Also because thedistributor is tabled tightly with the frit, and is normally alsorelatively tightly pressed in the groove of the column body, it isdifficult to remove it. If it is removed with force, the distributorcould often be damaged.

5. When packing having a small diameter is to be packed into achromatographic cake using the high pressure slurry method, possibleleakage must be tested before the cake can be packed because thediameter of a chromatographic cake is relatively great and, accordingly,it is difficult to seal it off if there is a leak. After thechromatographic packing is packed, if there is still a leakage problem,it can create great difficulties for repairing the apparatus.

6. For a relatively large chromatographic cake, a relatively largeslurry tank and associated devices are required which increase theproduction costs.

SUMMARY OF THE INVENTION

The principal object of this invention is to provide a kind of improvedchromatographic cake applicable to the separation, or renaturation withsimultaneous purification, of biopolymers with good results.

The present invention is directed to a type of chromatographic “cake,”which comprises a chromatographic packing cake or shell defining a fixedvolume inner region or cavity, and having a mobile phase inlet and amobile phase outlet, the chromatographic packing cake being packed inthe inner cavity thereof with a suitable chromatographic packingmaterial, wherein the ratio R of the thickness

In a preferred embodiment of this invention, the preferable dimensionsof the fixed volume inner cavity of the chromatographic packing cakerange from about 0.2–50 mm in thickness and from about 5.0–1000 mm indiameter.

A chromatographic packing cake in accordance with a preferred embodimentof this invention includes upper and lower clamp plates sandwiching acake body element, each clamp plate having either the said mobile phaseinlet or outlet respectively at opposite ends (clamp plates) of theapparatus, and also, preferably, wherein the cake body includes at leastone lateral hole or aperture.

In order to avoid the loss of chromatographic packing from the innercavity, frits are assembled or located at the lateral sides of the upperand lower clamp plates respectively of the chromatographic packing cakeand near to the inner cavity of the chromatographic packing cake. Thediameters of the frit holes in the frits should be greater than thetypical size of the biopolymers in the mobile phase but also less thanthe size of the chromatographic packing.

In order to obtain generally even distributions of mobile phase andsolute through the cake, and to enhance the separation effect, inanother preferred embodiment of the invention distributor elements areassembled or located between the upper and lower clamp plates of thechromatographic packing cake.

Such a distributor element comprises one generally flat plate havingsubstantially the same profile as the cross section of the inner cavityof the chromatographic packing cake. On the surface of at least onelateral side of each distributor element are radiating and concentriccircular blast grooves. Distributing holes are located at the junctionsof the radiating and circular guide grooves.

The preferred cross section of the guide grooves in the distributorelements is generally triangular and cambered.

The diameters of the distributing holes in the distributor elements mayincrease gradually as they move radially outward from the center of thedistributor element.

In another embodiment of chromatographic packing cakes in accordancewith this invention, a seal ring may be placed between the upper andlower clamp plates and the cake body.

Chromatographic cakes in accordance with this invention may beadvantageously used in the separation and purification of biopolymersprepared in biotechnology processes, thereby realizing advantages suchas high efficiency, processing of large volumes and good reproducibilityof results. The packed chromatographic cakes of this invention areequally useful under process conditions of either low or middlepressures. The cakes of this invention can decrease costs and enhanceoutput. With the chromatographic cakes of this invention, the processesof separation and purification, removal of denaturants during arenaturation process, coarse purification, and multiple-steppurifications can be combined and accomplished in only one step. Thechromatographic cakes of this invention can work under pressureconditions ranging from about 1–200 kg/cm².

A second objective of this invention is to provide new and improvedmanufacturing methods for preparing chromatographic cakes in accordancewith this invention, such methods being characterized by high speed, lowcost and high efficiency.

In order achieve this objective, this invention uses the followingtechnical outline of a manufacturing method for chromatographic cakes,the method including the following steps:

-   -   1. Manufacturing the chromatographic packing cake: The        chromatographic packing cake includes the upper and lower clamp        plates, having respectively either a mobile phase inlet or        outlet, assembled together to the cake body, which has at least        one lateral hole. The ratio of the thickness to the diameter of        the inner cavity of the chromatographic packing cake is smaller        than or equal to 1.    -   2. A suitable chromatographic packing is packed into the        assembled cake using the lateral hole(s) of the chromatographic        packing cake to insert the packing material into the inner        cavity of the chromatographic packing cake.

The lateral hole(s) of the chromatographic packing cake can be directlyconnected to the slurry tank of a high-pressure slurry packing machinefor packing the chromatographic cake.

When a chromatographic cake with a diameter of less than about 50 mm ismanufactured, the chromatographic cake can be directly placed on thehigh-pressure slurry packing machine. According to the usual method forpacking a column, the chromatographic packing can be directly added intothe inner cavity of the chromatographic packing cake.

When a chromatographic cake having a diameter greater than about 50 mmis manufactured, however, the cake should preferably be at leastpartially packed using a suction method in order to insure that thechromatographic packing are packed tightly. Using the suction method,the chromatographic cake is placed on the high-pressure slurry-packingmachine, and the chromatographic packing is added through the lateralhole(s) of the chromatographic packing cake into the inner cavity of thechromatographic packing cake.

The method of adding the chromatographic packing through the lateralhole of the chromatographic packing cake helps to reduce or eliminatethe “dead” volume in the chromatographic cake, which in turn improvesthe separation effect. Dead volume tends to increase by the loss ofchromatographic packing after the chromatographic cake has been in usefor some time. The method of adding additional chromatographic packingis similar to the original cake packing method.

In order to eliminate accumulated impurities on the distributorelements, the frits, and the biopolymers that do not enter the packedchromatographic cake, the chromatographic cake should be periodicallywashed through the lateral hole of the chromatographic packing cake.Because these accumulations can increase the pressure on thechromatographic system, they can result in contaminating follow-upseparated samples. A preferred washing method for the chromatographiccakes of this invention is to let buffer solution or water enter thelateral hole, which acts as an inlet, and go out from the inlet andoutlet respectively in the upper and lower clamp plates.

Another embodiment of this invention is to periodically replacedeteriorated chromatographic packing through the lateral hole of thechromatographic packing cake and to remove deteriorated chromatographicpacking which has a low column efficiency and which makes thechromatographic system pressure increase. The removed packing is thenreplaced with new packing. One such method is as follows: let the upperand lower clamp plates act as inlets and use the lateral hole as theoutlet; purge the deteriorated chromatographic packing with water, andrepack the new chromatographic packing into the hollow interior of thecake.

The operations for the cake packing and packing replacement inaccordance with this invention are very simple and easy, and they cansave time and workload. As the clamp plates on both ends of thechromatographic cake would not be opened after a leakage check, and thecake can be packed directly, this design can insure a chromatographiccake free from liquid leakage and having an even surface of packing,which is favorable to enhancing the column efficiency. When a biggerchromatographic cake is packed, it does not need a larger slurry tankand other accessory equipment, which means that production costs arealso reduced.

Further explanations of this invention in relation to the followingfigures and concrete examples appear below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic side sectional view of a chromatographic cake inaccordance with this invention.

FIG. 2 is a sectional view of the chromatographic cake of FIG. 1 takenalong the line A–A′.

FIG. 3 is a schematic top view of a distributor element for use with achromatographic cake in accordance with this invention.

FIG. 4 is a sectional view of the distributor element of FIG. 3 takenalong the line B–B′.

FIG. 5 is a chromatogram of a chromatographic cake in accordance withthis invention which has been radially packed.

FIG. 6 is a chromatogram of a chromatographic cake in accordance withthis invention which has been axially packed.

FIG. 7 is a chromatogram for a separation carried out in accordance withExample 5 below using a chromatographic cake in accordance with thisinvention.

FIG. 8 is a chromatogram for a separation carried out in accordance withExample 5 below using a chromatographic cake that has been washed andrepacked with additional packing in accordance with this invention.

FIG. 9 is a chromatogram for a separation carried out in accordance withExample 6 below using a chromatographic cake in accordance with thisinvention.

FIG. 10 is a chromatogram for a separation carried out in accordancewith Example 6 but using a conventional chromatographic column insteadof a chromatographic cake in accordance with this invention.

FIG. 11 is a chromatogram resulting from use of a 10×50 mm I.D.chromatographic cake in accordance with this invention to separate sevenstandard proteins.

FIG. 12 is a chromatogram resulting from use of a 10×200 mm I.D.chromatographic cake in accordance with this invention to separate fivestandard proteins.

FIG. 13 is a chromatogram resulting from the use of a 10×300 mm I.D.chromatographic cake in accordance with this invention to separate fivestandard proteins.

FIG. 14 is a chromatogram resulting from renaturation carried out withsimultaneous purification of rhINF-γ using a 10×50 mm I.D.chromatographic cake in accordance with this invention.

FIG. 15 schematically illustrates the multifunctional utility ofchromatographic cakes in accordance with this invention.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

Structure of a Chromatographic Cake in Accordance with this Invention

As shown in FIGS. 1 and 2, a chromatographic cake in accordance withthis invention preferably includes a stainless steel chromatographicpacking cake, as seen in FIG. 1, filled with a suitable chromatographicpacking 10 packed into the inner cavity of the chromatographic packingcake. The cross section of the inner cavity of the packing cake ispreferably generally round in shape, with a preferred thickness of about10 mm and having a diameter of about 100 mm. The degree of roughness onthe surface of the inner cavity of the chromatographic packing cake ispreferably smaller than about 1.6 μm so that it can be easily sealed andcan reduce the irreversible absorption of biopolymers.

A chromatographic packing cake in accordance with this inventioncomprises a pair of upper and lower clamp plates, 3 and 4 respectivelyin FIG. 1, with a mobile phase inlet 1 in one such clamp plate and amobile phase outlet 2 in the other such clamp plate, together with acake body 5 having at least one lateral hole or aperture 9 extendingfrom an external region into the inner cavity of the cake. The upper andlower clamp plates 3 and 4 and the cake body 5 define the inner cavityof the chromatographic packing cake. The lateral hole 9 through cakebody 5 is designed to be blocked and sealed with an end cap (not shown)after the chromatographic packing 10 has been packed into the innercavity. In order to prevent leakage, the seal rings 8, preferably madeof corrosion-resistant engineering plastic, are installed between theupper and lower clamp plates 3 and 4 respectively and cake body 5.Stainless steel frit elements 6 with holes (not seen in FIG. 1 becausethey are too small) are installed separately on the inner sides of theupper and lower clamp plates 3 and 4 adjacent the inner cavity of thechromatographic packing cake. The diameter of the frit 6 is greater thanthe diameter of the inner cavity of the chromatographic packing cake.The diameter of the frit holes is selected to be a size that is smallerthan the diameter of the particles of chromatographic packing 10 butlarger than the average size of typical biopolymers being processed.

As shown in FIGS. 3 and 4, the distributor elements 7 used in apreferred embodiment of this invention are preferably made ofengineering plastic, which is resistant to acid and alkali. Suchdistributor elements are installed between the upper and lower clampplates 3 and 4, respectively, and the associated frit 6. A distributorelement 7 comprises a plate member that has the same general shape asthe shape of the cross section of the inner cavity of thechromatographic packing cake. On the surfaces of one or both sides ofthe plate are both radiating and concentric circular blast grooves 11(same reference numeral used to identify both types of grooves). Thecross section of the blast grooves 11 is preferably of a generallytriangular shape. In the junctions between the radiating and circularblast grooves 11, are generally round distribution holes 12. In oneembodiment of this invention, the diameters of the distribution holesmay increase gradually as they are located further radially outward fromthe center of the distribution element. Such increasing size of thedistribution element holes 12 can be seen in FIG. 3.

In practice, the cross section of the inner cavity of a chromatographicpacking cake in accordance with this invention may have many differentshapes, for example, it can be round, polygonal, elliptic and so on.But, in each such design, the ratio of the thickness to the diameter (orcorresponding dimension) of the inner cavity of the chromatographicpacking cake should be smaller than or equal to 1. An optimal cakethickness has been found to be about 0.2–50 mm, with the correspondingdiametral dimension ranging from about 5.0–1000 mm. The chromatographicpacking cake can be made from many materials, such as resistant-to-acidand alkali stainless steel, titanium alloy, and many kinds ofengineering plastics. There are also many kinds of chromatographicpacking suitable for various types of chromatographic separations whichare useful with the chromatographic cakes of this invention. The numberof lateral holes 9 on the cake body 5 of the chromatographic packingcake may be one or more than one, which can be decided in accordancewith each actual situation.

EXAMPLE 2

Packing and Manufacturing a Medium-Larger Sized Chromatographic Cakewith a Diameter of more than 50 mm Using the Radial Column PackingMethod

-   -   1. A chromatographic cake was made in accordance with the method        described above in Example 1 and as illustrated in FIGS. 1–4;    -   2. The cake was at least partially packed using a usual suction        packing method; and,    -   3. The lateral hole(s) 9 of the chromatographic packing cake was        (were) connected directly with the slurry tank on a        high-pressure slurry-packing machine, and additional        chromatographic packing was added through the lateral hole(s) of        the chromatographic packing cake into the inner cavity of the        chromatographic packing cake to complete the packing step.

EXAMPLE 3

Packing and Manufacturing a Smaller-Sized Chromatographic Cake with aDiameter of less than 50 mm Using the Radial Column Packing Method

-   -   1. A chromatographic packing cake was made according to the        method described above in Example 1 and as illustrated in FIGS.        1–4; and,    -   2. The lateral hole(s) 9 of the chromatographic packing cake was        (were) connected directly with the slurry tank on a        high-pressure slurry-packing machine, and the chromatographic        packing was added through the lateral hole(s) of the        chromatographic packing cake into the inner cavity of the        chromatographic packing cake to fill the inner cavity.

EXAMPLE 4

Performance Comparison Between Two Methods of Packing a ChromatographicCake

Under the same cake packing conditions (40 MPa column/cake packingpressure and 30 min. column packing time), small particles (diameter ofabout 5 μm of a hydrophobic chromatographic packing were packedseparately into two identical 10×50 mm I.D. chromatographic cakes firstusing the usual axial column packing method and, second, using theradial column packing method of this invention. Under the samechromatographic conditions (flow rate of 5.0 mL/min and gradient of0–100% B for 25 min.), five proteins (cytochrome C, ribonuclease A,lysozyme, α-amylase and insulin) were separated using the two packedcakes. The above operation was repeated five times for each cake. Thechromatograms showing the results of the two packing methods arerespectively shown in FIGS. 5 and 6. In the Figures, peak 1=cytochromoidC, peak 2=ribonuclease A, peak 3=lysozyme, peak 4=α-amylase and peak5=insulin. Comparing FIGS. 5 and 6, it can be seen from the results inthe Figures that good, substantially similar separation results areobtained with both of the column packing methods for the standardprotein separation. One difference found with this Example was that theradial packing method had somewhat better reproducibility of resultsthan the axial column packing method.

EXAMPLE 5

Separation Performance After Radially Discharging and Repacking aChromatographic Cake

A hydrophobic chromatographic packing was packed through the lateralhole of a chromatographic cake using the radial high pressure slurrymethod, after which the lateral hole was sealed with an end cap. Underthe conditions of a flow rate of 5 m L/min and a gradient of 100% A–100%B for a period of 25 mins., cytochrome C, myoglobin, lysozyme andα-amylase were separated. The results are shown in FIG. 7. In FIG. 7,peak 1=cytochrome C, peak 2=ribonuclease A, peak 3=lysozyme, and peak4=α-amylase. Following this separation, the chromatographic cake waswashed thoroughly. The lateral hole was used as a wash fluid outlet, andthe inlet and outlet of the clamp plates of the chromatographic cakewere used as wash fluid inlets, using water as the mobile phase. Achromatographic pump was turned on to assist with purging the usedpacking. Packing was added to replace deteriorated packing and any lostduring the wash step. After the packing was further processed throughdegassing with up sonic and evenly slurried, the chromatographic cakewas repacked using the same column packing method as previously. Underthe same chromatographic conditions used previously, the four proteinswere again successfully separated using the washed and repackedchromatographic cake. The results are shown in FIG. 8, in which peak1=cytochrome C, peak 2=ribonuclease A, peak 3=lysozyme, and peak4=α-amylase. From a comparison of FIGS. 7 and 8, no significantdifference was seen comparing the original separation with theseparation carried out after the original packing had been dischargedfrom the lateral hole of the chromatographic cake, washed, treated andrepacked in the chromatographic cake again without removal of the upperand lower clamp plates. This Example shows that it is very easy todischarge used, deteriorated packing through the lateral hole of thechromatographic cake, and then also easy to repack the cake with washedor new packing or to add additional packing.

EXAMPLE 6

Comparison of Separations Carried out Using a Chromatographic Column anda Chromatographic Cake

A chromatographic cake with the specification of 5×50 mm I.D. and achromatographic column with the specification of 200×7.9 mm I.D. wereselected for the Example. The volumes of the packing cavities in bothcases was 9.9±0.2 mL. Under the same 40 MPa pressure conditions, bothchromatographic apparatuses were packed using the same batch of HPHICpacking. Under the conditions of same sample size and flow rate of 4.0ml/min., six standard proteins were separated using the twochromatographic devices. The results are shown in FIGS. 9 and 10,respectively, in which peak 1=cytochrome C, peak 2=myoglobin, peak3=ribonuclease A, peak 4=lysozyme, peak 5=α-amylase, and peak 6=insulin.It can be seen from comparing FIGS. 9 and 10 that the chromatographiccake and the chromatographic column produce generally comparableresolutions for the six proteins. But, the advantage of thechromatographic cake according to this invention is that the thicknessof the chromatographic cake is only 1/40 the length of thechromatographic column with substantially the same geometric volumes ofthe respective packed beds. This Example thus shows that satisfactorychromatographic resolution is achieved with the chromatographic cakes ofthis invention even though they are configured with a relatively largerdiameter and relatively short column length, but with the same geometricvolume as a conventional chromatographic column.

EXAMPLE 7

Separations Using Different Types of Chromatographic Cakes

In this Example, the standard proteins were separated using three sizesof chromatographic cakes manufactured in accordance with this invention,namely a 10×50 mm I.D. cake, a 10×200 mm I.D. cake and a 10×300 mm I.D.cake. The results are shown respectively in FIGS. 11, 12 and 13. Asshown in FIG. 11 wherein peak 1=cytochrome C, peak 2=myoglobin, peak3=ribonuclease A, peak 4=lysozyme, peak 5=α-chmotropsen, peak6=α-amylase, and peak 7=insulin, the proteins are separated under thechromatographic conditions of 5.0 mL/min. flow rate, 0.08 AUFS, and agradient ranging from 100% A to 100% B for a period of 40 mins. As shownin FIG. 12, wherein peak 1=cytochrome C, peak 2=myoglobin, peak3=lysozyme, peak 4=α-amylase, and peak 5=insulin, the proteins areseparated under the chromatographic conditions of 100.0 ml/min. flowrate, 0.05 AUFS, and a gradient ranging from 100% A to 100% B for aperiod of 40 mins. As shown in FIG. 13, wherein peak 1=cytochrome C,peak 2=myoglobin, peak 3=ribonuclease A, peak 4=lysozyme, and peak5=α-amylase, the proteins are separated under the chromatographicconditions of 120.0 ml/min. flow rate, 0.1 AUFS, and a gradient rangingfrom 100% A to 100% B for a period of 60 mins. It can be seen in theFigures that all of these chromatographic cakes with differentspecifications result in satisfactory resolution.

EXAMPLE 8

Renaturation Efficiency of Denatured Lysozyme with Urea and GuanidinineHydrochloride Using a Chromatographic Cake

In this Example, under two different chromatographic conditions, sampleinjections were made of denatured lysozyme into solutions of urea andguanidinine hydrochloride (GuHCl), after the 5×50 mm I.D.chromatographic cakes in accordance with this invention wereequilibrated with solution A. Then the renatured components coming fromthe chromatographic cakes were collected. The bioactivity recovery ofthese effluent streams was measured as shown in Table 1 below. Thegradient used was changed from 100% A to 100% B. It can be seen from theresults in Table 1 that the chromatographic cake has a substantialrenaturation effect on the denatured lysozyme by urea and GuHCl.

TABLE 1 Bioactivity recovery of Lysozyme under different chromatographicconditions Flow rate, 4 mL/min. Flow rate, 2. mL/min. 25 min. linear 50min. linear Sample gradient time gradient time Denatured Lysozyme by102.9% 104.7% urea Denatured Lysozyme by 103.7% 104.0% GuHCl

EXAMPLE 9

Renaturation with Simultaneous Purification of the Recombined HumanInterferon rhINF-γ Using a Chromatographic Cake

FIG. 14 shows the chromatographic results of this Example in whichrenaturation is carried out simultaneously with purification of rhINF-γin a 10×50 mm I.D. chromatographic cake in accordance with thisinvention. The sample used was an rhINF-γ solution extracted from thecellular cataclastic solution of E. Coli with 7.0 mol/L GuHCl solution.The bioactivity measuring method of rhINF-γ was the restraint method ofcellular pathologic change. The operating conditions were as follows:the 7.0 mol/L GuHCl solution containing rhINF-γ of 1 mL was injectedinto the chromatographic cake equilibrated with mobile phase A from theextracting solution of E. coli at a flow rate of about 3.0–7.0 mL/min.over an interval of 25–40 mins., wherein the composition of the mobilephase was changed gradually from 100% A to 100% B. The fractions werecollected and their bioactivities were measured separately. It can beseen in the results that the bioactivity recovery was 1774.57%, i.e., 17times greater than the results achieved using the usual methods.

Industrial Applications

Chromatographic cakes in accordance with this invention can be usedsuccessfully at a pressure of more than 20 Mpa. Such cakes do not deformduring use, which helps to insure the surface evenness of thechromatographic packing at the end of a sample injection which, in turn,enhances the resolution. Using the cakes of this invention, separationis fast even with a low system pressure (e.g., lower than 5.0 Mpagenerally). From the standpoint of good resolution under high flow rateconditions, the performance of chromatography cakes in accordance withthis invention is comparable to that of perfusion chromatography and,therefore, has superior industrial applicability. The chromatographiccakes of this invention also allow for sample injections having arelatively high viscosity and a little precipitation. When thechromatographic separation is completed with the cakes of thisinvention, no fixed inlet and outlet are needed. The cakes of thisinvention show only a small irreversible absorption on objectiveproducts, which enhances the recovery of such objective products. Withthe chromatographic cake of this invention, the separation,renaturation, and purification processes can be performed in one step,which makes it at least three times simpler than the normal renaturationand purification technology. With the chromatographic cakes of thisinvention, the relevant production period can also be shortened by atleast several times compared with comparable conventional processes andchromatographic devices. The investment for equipment is also reducedsignificantly using the chromatographic cakes of this invention. At thesame time, denaturants can also be recovered when using chromatographiccakes in accordance with this invention. Not only can such recovereddenaturants be reused, but also the environmental pollution caused bydisposing of such denaturants can be reduced. FIG. 15 shows that achromatographic cake in accordance with this invention can play a rollof “killing four birds with one stone”, (that is, quick and completeelimination of denaturants, easy recycling of denaturants, proteinrenaturation, and separation of impure proteins). Using only the samepacking volume as a conventional chromatographic column, in comparisonwith normal columns, more packing material can be packed into the innercavity of a chromatographic cake packed in accordance with thisinvention. Relatively greater mass loading and volume loading istherefore possible with the chromatographic cakes of this invention.

Thus, the chromatographic cakes of this invention will find a wide rangeof applications in the separation, renaturation and purification ofbiopolymers.

While the present invention has been particularly shown and describedabove with reference to various exemplary embodiments thereof, it willbe understood by those of ordinary skill in the art that various changesin form and details may be made therein without departing from thespirit and scope of the present invention, as defined by the followingclaims.

1. Chromatographic cake apparatus comprising in combination: a tubularbody section open-ended at both ends defining a central axis and havingat least a lateral opening therein, said lateral opening extendingbetween an outer face of the body section and an inner face of the bodysection in a lateral direction that is generally orthogonal to thecentral axis; first and second clamp plates fastened respectively atfirst and second ends of the tubular body section so as to define afixed volume inner cavity of said apparatus having a cavity thickness asmeasured along said central axis that ranges from about 0.2–50 mm and alateral dimension of said cavity measured along said lateral directionthat ranges from about 5.0–1000 mm, wherein the ratio of cavitythickness to lateral dimension does not exceed 0.20; each of said firstand second clamp plates having inner and outer clamp plate sides and amobile phase inlet or outlet aperture along said central axis extendingbetween the inner and outer clamp plate sides, said inlet or outletaperture being capable of passing a mobile phase through a centralportion of said clamp plate respectively into or out of said innercavity; and, first and second frit elements positioned on the innerclamp plate sides adjacent an end of said inner cavity, each fritelement comprising frit holes of a frit hole size so as to retainparticulate chromatographic material having an average particle sizegreater than the frit hole size inside the inner cavity, whilepermitting passage of a mobile phase containing a material sized to passthrough the frit holes.
 2. A chromatographic cake apparatus according toclaim 1 further comprising first and second distributor elements, eachpositioned between a frit element and its associated clamp plate, saiddistributor elements comprising suitably sized plate members having onat least one face thereof multiple radiating grooves intersectingmultiple concentric circular grooves wherein the junctions between theradiating grooves and the circular grooves are mobile phase distributionholes.
 3. A chromatographic cake apparatus according to claim 2, furtherwherein the diameters of the mobile phase distribution holes graduallyincrease as they are located further radially outward from the center ofthe distributor element.
 4. A chromatographic cake apparatus accordingto claim 1, further wherein the inner cavity is generally round in shapewith a thickness of about 10 mm and having a diameter in the range ofabout 50 mm to 300 mm.
 5. A chromatographic cake apparatus according toclaim 1 further comprising first and second seal rings, each positionedbetween an end of the tubular body section and the associated clampplate, effective to seal the inner cavity except at said mobile phaseinlet and outlet apertures and at said lateral opening.
 6. Achromatographic cake apparatus according to claim 1 wherein said bodysection, said clamp plates, and said frit elements are made of stainlesssteel to accommodate operational pressures in the range of 1–200 kg/cm².7. A chromatographic cake apparatus according to claim 1 wherein thecavity thickness ranges from about 0.2 to 50 mm, the correspondinglateral dimension ranges from about 5.0 to 1000 mm, and the ratio of thecavity thickness to lateral dimension is about 0.20 to 0.033.
 8. Achromatographic cake apparatus according to claim 1 wherein said tubularbody section has a cross-sectional shape selected from round, polygonal,or elliptical.
 9. A chromatographic cake apparatus according to claim 1wherein said tubular body section has a cross-sectional shape selectedfrom round, polygonal, or elliptical.
 10. Chromatographic cake apparatuscomprising in combination: a tubular body section open-ended at bothends defining a central axis and having at least a lateral openingtherein, said lateral opening extending between an outer face of thebody section and an inner face of the body section in a lateraldirection that is generally orthogonal to the central axis; first andsecond clamp plates fastened respectively at first and second ends ofthe tubular body section so as to define a fixed volume inner cavity ofsaid apparatus having a cavity thickness as measured along said centralaxis that ranges from about 0.2–50 mm and a lateral dimension of saidcavity measured along said lateral direction that ranges from about5.0–1000 mm, wherein the ratio of cavity thickness to lateral dimensiondoes not exceed 0.20; each of said first and second clamp plates havinginner and outer clamp plate sides and a mobile phase inlet or outletaperture along said central axis extending between the inner and outerclamp plate sides, said inlet or outlet aperture being capable ofpassing a mobile phase through a central portion of said clamp platerespectively into or out of said inner cavity; first and second fritelements positioned on the inner clamp plate sides adjacent an end ofsaid inner cavity, each frit element comprising frit holes of a frithole size so as to retain particulate chromatographic material having anaverage particle size greater than the frit hole size inside the innercavity, while permitting passage of a mobile phase containing a materialsized to pass through the frit holes; an end cap sealing said lateralopening; and, a chromatographic packing material having an averageparticle size that is larger than the frit hole size packed into saidinner cavity.
 11. A chromatographic cake apparatus according to claim 10further comprising first and second distributor elements, eachpositioned between a frit element and its associated clamp plate, saiddistributor elements comprising suitably sized plate members having onat least one face thereof multiple radiating grooves intersectingmultiple concentric circular grooves wherein the junctions between theradiating grooves and the circular grooves are mobile phase distributionholes.
 12. A chromatographic cake apparatus according to claim 10,further wherein the diameters of the mobile phase distribution holesgradually increase as they are located further radially outward from thecenter of the distributor element.
 13. A chromatographic cake apparatusaccording to claim 10, further wherein the inner cavity is generallyround in shape with a thickness of about 10 mm and having a diameter inthe range of about 50 mm to 300 mm.
 14. A chromatographic cake apparatusaccording to claim 10 further comprising first and second seal rings,each positioned between an end of the tubular body section and theassociated clamp plate, effective to seal the inner cavity except atsaid mobile phase inlet and outlet apertures and at said lateralopening.
 15. A chromatographic cake apparatus according to claim 10wherein said body section, said clamp plates, and said frit elements aremade of stainless steel.
 16. A chromatographic cake apparatus accordingto claim 10 wherein the cavity thickness ranges from about 0.2 to 50 mm,the corresponding lateral dimension ranges from about 5.0 to 1000 mm,and the ratio of the cavity thickness to lateral dimension is about 0.20to 0.033.
 17. Chromatographic cake apparatus comprising in combination:a tubular body section open-ended at both ends defining a central axisand having at least a lateral opening therein, said lateral openingextending between an outer face of the body section and an inner face ofthe body section in a lateral direction that is generally orthogonal tothe central axis; first and second clamp plates fastened respectively atfirst and second ends of the tubular body section so as to define afixed volume inner cavity of said apparatus having a cavity thickness asmeasured along said central axis that is about 0.20 to 0.033 time thelateral dimension of said cavity measured along said lateral direction;each of said first and second clamp plates having inner and outer clampplate sides and a mobile phase inlet or outlet aperture along saidcentral axis extending between the inner and outer clamp plate sides,said inlet or outlet aperture being capable of passing a mobile phasethrough a central portion of said clamp plate respectively into or outof said inner cavity; and, first and second frit elements positioned onthe inner clamp plate sides adjacent an end of and said inner cavity,each frit element comprising frit holes of a frit hole size so as toretain particulate chromatographic material having an average particlesize greater than the frit hole size inside the inner cavity, whilepermitting passage of a mobile phase containing a material sized to passthrough the frit holes.